Over the past decade evidence has accumulated strongly suggesting that astrocytes participate in synaptic transmission through their release of gliotransmitters, leading to the concept of the tripartite synapse. Findings from our laboratory as well as a number of other laboratories indicate that increases in astrocytic Ca2+ in situ lead to the release of glutamate that can activate both metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs) on pre- and postsynaptic neuronal membranes. Further, in a Ca2+- dependent manner, astrocytes appear to release ATP that is converted to adenosine which then modulates synaptic transmission through the activation of presynaptic adenosine receptors. Through their release of glutamate and ATP/adenosine astrocytes have been reported to affect processes ranging from synaptic plasticity to vascular tone. Most of these studies used either receptor ligands thought to selectively activate astrocytic Gq-coupled GPCRs or caged IP3/Ca2+ to increase astrocytic Ca2+. In an effort to selectively study the role of astrocytic G-protein coupled receptors (GPCRs), we have made a series of transgenic mouse lines expressing GPCRs that 1) are not expressed by other cells in brain, 2) are not activated by ligands released in brain, and 3) whose ligand does not activate endogenous GPCRs present in brain. We fully anticipated that activating a Gq-linked GPCR definitively localized to astrocytes would lead to effects similar to those reported by ourselves and others using caged Ca2+ or caged IP3 to increase Ca2+ in astrocytes; we were wrong. As recently reported in Neuron, we have very strong evidence that activating Gq-linked GPCRs that lead to Ca2+ increases in most astrocytes does not lead to the release of gliotransmitters that affect the activity of CA1 pyramidal neurons. This finding brings into question a great number of high profile publications (including our own) that astrocytes release gliotransmitters following increases in Ca2+. In the first Specific Aim we propose to carry out a series of experiments to clarify the mechanisms controlling the release of gliotransmitters following GPCR-mediated increases in astrocytic Ca2+. Most attention in this field has focused on Ca2+-dependent signaling in astrocytes. However, it is very likely that GPCRs linked to cyclic AMP are also playing an important role in neurophysiology. In spite of the fact that all astrocytes examined exhibit GPCRs linked to cyclic AMP formation, we know virtually nothing concerning the role of astrocytic Gs-coupled GPCRs in neurophysiology or neurological disorders. In contrast, the activation of Ca2+ signaling in astrocytes is thought to affect a wide variety of processes ranging from LTP and vascular tone to inflammation and chronic pain. In the second Specific Aim, we will take advantage of our ability to specifically disrupt GPCR-mediated increases in astrocytic Ca2+ and cyclic AMP via conditional gene knockouts (cKOs). In these experiments we will examine the role of astrocytic Ca2+ and cyclic AMP signaling cascades in processes reported to be modulated by astrocytes. In the third Specific Aim, we will investigate the role of astrocytic Gs- and Gq-GPCR-dependent signaling cascades in behavior. Virtually nothing is known concerning the role of astrocytic GPCRs in behavior or neurological disorders. PUBLIC HEALTH RELEVANCE: Astrocytes are the most numerous cells in brain (~50% of all brain cells) and associate with all cellular elements; a single astrocyte can envelop as many as a 100,000 synapses. Over the last decade a large number of reports suggest that astrocytes participate in synaptic transmission by releasing gliotransmitters that modulate, and even drive, synaptic transmission. Most of these studies were carried out using non physiological approaches to selectively activate astrocytic G-protein coupled receptors (GPCRs). To take a more physiological approach, we have developed a number of genetically modified mice that enable us to either selectively stimulate or block GPCR-mediated signaling cascades in astrocytes. Results obtained using these new model systems do not support the previous literature (including our own) in this area. The goal of this proposal is to take a genetic approach to investigate the role of astrocytes in a wide array of physiological and behavioral processes.